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Yellow Fluorescent Protein (yellow + fluorescent_protein)
Selected AbstractsImpaired nerve regeneration in reeler mice after peripheral nerve injuryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2008Erika Lorenzetto Abstract Reelin, an extracellular matrix protein, plays an important role in the regulation of neuronal migration and cortical lamination in the developing brain. Little is known, however, about the role of this protein in axonal regeneration. We have previously shown that Reelin is secreted by Schwann cells in the peripheral nerve compartment during postnatal development and that it is up-regulated following nerve injury in adult mice. In this work, we generated mice deficient in Reelin (reeler) that express yellow fluorescent protein (YFP) in a subset of neurons and examined the axonal regeneration following nerve crush. We found that axonal regeneration was significantly altered compared with wild-type mice. By contrast, retrograde tracing with Fluorogold dye after sciatic nerve crush was unaffected in these mutants, being comparable with normal axonal transport observed in wild-type. These results indicate that the absence of Reelin impairs axonal regeneration following injury and support a role for this protein in the process of peripheral nerve regeneration. [source] Heterogeneity of V2-derived interneurons in the adult mouse spinal cordEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2007A. Al-Mosawie Abstract Spinal neurons and networks that generate rhythmic locomotor activity remain incompletely defined, prompting the use of molecular biological strategies to label populations of neurons in the postnatal mouse. During spinal cord development, expression of Lhx3 in the absence of Isl1 specifies a V2 interneuronal fate. In this study, postnatal V2-derived interneurons were identified by yellow fluorescent protein (YFP) expression in the double-transgenic offspring of Lhx3Cre/+ × thy1-loxP-stop-loxP-YFP mice. While some motoneurons were labelled, several populations of interneurons predominantly located in lamina VII could also be distinguished. Small interneurons were located throughout the spinal cord whereas larger interneurons were concentrated in the lumbar enlargement. Some V2-derived interneurons were propriospinal, with axons that bifurcated in the lateral funiculus. V2-derived interneurons gave rise to populations of both excitatory and inhibitory interneurons in approximately equal proportions, as demonstrated by in situ hybridization with VGLUT2 mRNA. Immunohistochemical studies revealed YFP+ boutons throughout the spinal cord. Both glutamatergic and glycinergic YFP+ boutons were observed in lamina IX where many apposed motoneuron somata. GABAergic YFP+ boutons were also observed in lamina IX, and they did not form P-boutons. At P0, more than half of the YFP+ interneurons expressed Chx10 and thus were derived from the V2a subclass. In adult mice, there was an increase in Fos expression in V2-derived interneurons following locomotion, indicating that these neurons are active during this behaviour. The heterogeneity of V2-derived interneurons in adult mice indicates that physiologically distinct subpopulations, including last-order interneurons, arise from these embryonically defined neurons. [source] Estrogen modulates neuronal movements within the developing preoptic area,anterior hypothalamusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2007John Gabriel Knoll Abstract The preoptic area,anterior hypothalamus (POA-AH) is characterized by sexually dimorphic features in a number of vertebrates and is a key region of the forebrain for regulating physiological responses and sexual behaviours. Using live-cell fluorescence video microscopy with organotypic brain slices, the current study examined sex differences in the movement characteristics of neurons expressing yellow fluorescent protein (YFP) driven by the Thy-1 promoter. Cells in slices from embryonic day 14 (E14), but not E13, mice displayed significant sex differences in their basal neuronal movement characteristics. Exposure to 10 nm estradiol-17, (E2), but not 100 nm dihydrotestosterone, significantly altered cell movement characteristics within minutes of exposure, in a location-specific manner. E2 treatment decreased the rate of motion of cells located in the dorsal POA-AH but increased the frequency of movement in cells located more ventrally. These effects were consistent across age and sex. To further determine whether early-developing sex differences in the POA-AH depend upon gonadal steroids, we examined cell positions in mice with a disruption of the steroidogenic factor-1 gene, in which gonads do not form. An early-born cohort of cells were labelled with the mitotic indicator bromodeoxyuridine (BrdU) on E11. More cells were found in the POA-AH of females than males on the day of birth (P0) regardless of gonadal status. These results support the hypothesis that estrogen partially contributes to brain sexual dimorphism through its influence on cell movements during development. Estrogen's influence may be superimposed upon a pre-existing genetic bias. [source] Characterization of signaling pathway for the translocation of neuronal nitric oxide synthase to the plasma membrane by PACAPJOURNAL OF NEUROCHEMISTRY, Issue 6 2008Takayuki Ohnishi Abstract In the central nervous system, the activation of neuronal nitric oxide synthase (nNOS) is closely associated with activation of NMDA receptor, and trafficking of nNOS may be a prerequisite for efficient NO production at synapses. We recently demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) and NMDA synergistically caused the translocation of nNOS to the membrane and stimulated NO production in PC12 (pheochromocytoma) cells. However, the mechanisms responsible for trafficking and activation of nNOS are largely unknown. To address these issues, here we constructed a yellow fluorescent protein (YFP)-tagged nNOS N-terminal (1,299 a.a.) mutant, nNOSNT-YFP, and visualized its translocation in PC12 cells stably expressing it. PACAP enhanced the translocation synergistically with NMDA in a time- and concentration-dependent manner. The translocation was blocked by inhibitors of protein kinase A (PKA), protein kinase C (PKC), and Src kinase; and the effect of PACAP could be replaced with PKA and PKC activators. The ,-finger region in the PSD-95/disc large/zonula occludens-1 domain of nNOS was required for the translocation of nNOS and its interaction with post-synaptic density-95 (PSD-95), and NO formation was attenuated by dominant negative nNOSNT-YFP. These results demonstrate that PACAP stimulated nNOS translocation mediated by PKA and PKC via PAC1 -receptor (a PACAP receptor) and suggest cross-talk between PACAP and NMDA for nNOS activation by Src-dependent phosphorylation of NMDA receptors. [source] Nitric oxide inhibits mitochondrial movement in forebrain neurons associated with disruption of mitochondrial membrane potentialJOURNAL OF NEUROCHEMISTRY, Issue 3 2006Gordon L. Rintoul Abstract Nitric oxide (NO) has a number of physiological and pathophysiological effects in the nervous system. One target of NO is the mitochondrion, where it inhibits respiration and ATP synthesis, which may contribute to NO-mediated neuronal injury. Our recent studies suggested that impaired mitochondrial function impairs mitochondrial trafficking, which could also contribute to neuronal injury. Here, we studied the effects of NO on mitochondrial movement and morphology in primary cultures of forebrain neurons using a mitochondrially targeted enhanced yellow fluorescent protein. NO produced by two NO donors, papa non-oate and diethylamine/NO complex, caused a rapid cessation of mitochondrial movement but did not alter morphology. Movement recovered after removal of NO. The effects of NO on movement were associated with dissipation of the mitochondrial membrane potential. Increasing cGMP levels using 8-bromoguanosine 3,,5,-cyclic monophosphate, did not mimic the effects on mitochondrial movement. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-induced activation of soluble guanylate cyclase, did not block the effects of NO. Thus, neither increasing nor decreasing cGMP levels had an effect on mitochondrial movement. Based on these data, we conclude that NO is a novel modulator of mitochondrial trafficking in neurons, which may act through the inhibition of mitochondrial function. [source] Neurovascular Alignment in Adult Mouse Skeletal MusclesMICROCIRCULATION, Issue 2 2005SHAWN E. BEARDEN ABSTRACT Objective: Muscle blood flow increases with motor unit recruitment. The physical relationships between somatic motor nerves, which control muscle fiber contraction, and arterioles, which control microvascular perfusion, are unexplored. The authors tested the hypothesis that motor axons align with arterioles in adult skeletal muscle. Methods: Transgenic mice (C57BL/6 background, n = 5; 10 months of age) expressing yellow fluorescent protein in all motor nerves underwent vascular casting (Microfil). Excised epitrochlearis, gracilis, gluteus maximus, and spinotrapezius muscles were imaged at 380× and 760× and a computer-integrated tracing system (Neurolucida) was used to acquire 3-dimensional digital renderings of entire arteriolar and neural networks within each muscle. Results: Arteriolar networks were typically ,3-fold longer than neural networks. Nerves coursed with arterioles until terminating at motor endplates. Across muscles, proximity analyses revealed that , 75% of total nerve length (9.8,48.8 mm) lay within 200 ,m of the nearest arteriole (diameters of 15,60 , m). Conclusions: Somatic motor nerves and arterioles align closely within adult mammalian skeletal muscle. Understanding the signals governing neurovascular alignment may hold important clues for the advancement of tissue engineering and regeneration. [source] The ,-1,3-glucanosyltransferase gas4p is essential for ascospore wall maturation and spore viability in Schizosaccharomyces pombeMOLECULAR MICROBIOLOGY, Issue 5 2008María De Medina-Redondo Summary Meiosis is the developmental programme by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. The formation of the Schizosaccharomyces pombe ascospore wall requires the co-ordinated activity of enzymes involved in the biosynthesis and modification of its components, such as glucans. During sporogenesis, the ,-1,3-glucan synthase bgs2p synthesizes linear ,-1,3-glucans, which remain unorganized and alkali-soluble until covalent linkages are set up between ,-1,3-glucans and other cell wall components. Several proteins belonging to the glycoside hydrolase family 72 (GH72) with ,-1,3-glucanosyltransferase activity have been described in other organisms, such as the Saccharomyces cerevisiae Gas1p or the Aspergillus fumigatus Gel1p. Here we describe the characterization of gas4+, a new gene that encodes a protein of the GH72 family. Deletion of this gene does not lead to any apparent defect during vegetative growth, but homozygous gas4, diploids show a sporulation defect. Although meiosis occurs normally, ascospores are unable to mature or to germinate. The expression of gas4+ is strongly induced during sporulation and a yellow fluorescent protein (YFP),gas4p fusion protein localizes to the ascospore periphery during sporulation. We conclude that gas4p is required for ascospore maturation in S. pombe. [source] Two different Pseudomonas aeruginosa chemosensory signal transduction complexes localize to cell poles and form and remould in stationary phaseMOLECULAR MICROBIOLOGY, Issue 1 2006Zehra Tüzün Güvener Summary Pseudomonas aeruginosa has sets of sensory genes designated che and che2. The che genes are required for flagella-mediated chemotaxis. The che2 genes are expressed in the stationary phase of growth and are probably also involved in flagella,mediated behavioural responses. P. aeruginosa also has 26 chemoreceptor genes, six of which are preferentially expressed in stationary phase. Subcellular localization experiments indicated that Che proteins form signal transduction complexes at cell poles throughout growth. Cyan fluorescent protein (CFP)-tagged McpA, a stationary phase-expressed chemoreceptor, appeared and colocalized with yellow fluorescent protein (YFP)-tagged CheA when cells entered stationary phase. This indicates that P. aeruginosa chemotaxis protein complexes are subject to remoulding by chemoreceptor proteins that are expressed when cells stop growing. CheA-CFP and CheY2-YFP tagged proteins that were coexpressed in the same cell had separate subcellular locations, indicating that Che2 proteins do not enter into direct physical interactions with Che proteins. Che2 protein complex formation required McpB, another stationary phase induced chemoreceptor that is predicted to be soluble. This implies that Che2 complexes have a function that depends on just one chemoreceptor. Our results suggest that motile P. aeruginosa cells have signal transduction systems that are adapted to allow non-growing cells to sense and respond to their environment differently from actively growing cells. [source] FtsQ, FtsL and FtsI require FtsK, but not FtsN, for co-localization with FtsZ during Escherichia coli cell divisionMOLECULAR MICROBIOLOGY, Issue 2 2001Joseph C. Chen During cell division in Gram-negative bacteria, the cell envelope invaginates and constricts at the septum, eventually severing the cell into two compartments, and separating the replicated genetic materials. In Escherichia coli, at least nine essential gene products participate directly in septum formation: FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ and ZipA. All nine proteins have been localized to the septal ring, an equatorial ring structure at the division site. We used translational fusions to green fluorescent protein (GFP) to demonstrate that FtsQ, FtsL and FtsI localize to potential division sites in filamentous cells depleted of FtsN, but not in those depleted of FtsK. We also constructed translational fusions of FtsZ, FtsA, FtsQ, FtsL and FtsI to enhanced cyan or yellow fluorescent protein (ECFP or EYFP respectively), GFP variants with different fluorescence spectra. Examination of cells expressing different combinations of the fusions indicated that FtsA, FtsQ, FtsL and FtsI co-localize with FtsZ in filaments depleted of FtsN. These localization results support the model that E. coli cell division proteins assemble sequentially as a multimeric complex at the division site: first FtsZ, then FtsA and ZipA independently of each other, followed successively by FtsK, FtsQ, FtsL, FtsW, FtsI and FtsN. [source] Functional Expression, Targeting and Ca2+ Signaling of a Mouse Melanopsin-eYFP Fusion Protein in a Retinal Pigment Epithelium Cell Line,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Maikel E. Giesbers Melanopsin, first discovered in Xenopus melanophores, is now established as a functional sensory photopigment of the intrinsically photosensitive retinal ganglion cells. These ganglion cells drive circadian rhythm and pupillary adjustments through projection to the brain. Melanopsin shares structural similarities with all known opsins. Comprehensive characterization of melanopsin with respect to its spectral properties, photochemical cascade and signaling partners requires a suitable recombinant system and high expression levels. This combination has not yet been described. To address this issue, we have expressed recombinant mouse melanopsin in several cell lines. Using enhanced yellow fluorescent protein (eYFP) as a visualization tag, expression was observed in all cell lines. Confocal microscopy revealed that melanopsin was properly routed to the plasma membrane only in retinal pigment epithelium (RPE)-derived D407 cells and in human embryonic kidney (HEK) cells. Further, we performed intracellular calcium measurements in order to probe the melanopsin signaling activity of this fusion protein. Transfected cells were loaded with the calcium indicator Fura2-AM. Upon illumination, an immediate but transient calcium response was observed in HEK as well as in D407 cells, while mock-transfected cells showed no calcium response under identical conditions. Supplementation with 11- cis retinal or all- trans retinal enhanced the response. After prolonged illumination the cells became desensitized. Thus, RPE-derived cells expressing recombinant melanopsin may constitute a suitable system for the study of the structural and functional characteristics of melanopsin. [source] Detection of transient protein,protein interactions by bimolecular fluorescence complementation: The Abl-SH3 casePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2007Montse Morell Abstract Protein,protein interactions are essential in most biological processes. Many proteomic approaches have succeeded in the identification of strong and obligatory interactions but the study of weak and transient protein,protein interactions is still a challenge. The aim of the present study was to test the ability of bimolecular fluorescence complementation to detect and discriminate in vivo weak intracellular protein interactions. As a test case, the interaction of the SH3 domain from the c-Abl tyrosine kinase with both natural and designed targets has been chosen. The reassociation of functional yellow fluorescent protein (YFP) from its fragments requires previous binding between the SH3 domain and its partners; but once this occurs, the complex is trapped, turning transient SH3 interactions into stable, easily detectable ones. The method is very sensitive and can be implemented for proteomic analysis of weak protein interactions using flow cytometry. The fluorescence emission is dependent on the strength of the interaction, in such a way that it can be used, at least qualitatively, to screen for best binding candidates among similar proline-rich peptides. In addition, it is illustrated how this method can be used to gain structural insights into particular c-Abl SH3 interactions. [source] A Proteomics Approach to Cloning Fenestrin from the Nuclear Exchange Junction of TetrahymenaTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 4 2008ERIC S. COLE ABSTRACT. We set out to find the "fenestrin" gene, a gene whose protein is associated with numerous cellular apertures, including the nuclear exchange junction in mating Tetrahymena thermophila. First we developed protocols for imaging and isolating intact nuclear exchange junctions from conjugating cells. Proteins from these junctions were purified using SDS-PAGE, subjected to limited proteolysis, and precise molecular weights were determined by mass spectrometry. Using Protein Prospector® software and the published Tetrahymena Genome Database, genes for 15 of the most abundant proteins found in our extracts were identified. The most promising candidate was cloned by PCR, fused to yellow fluorescent protein (YFP), and placed under the control of an inducible metallothionein promoter. YFP-localization within live Tetrahymena transformants strongly suggested that one of these genes encoded the fenestrin protein, a result that was subsequently confirmed by Western blotting. [source] Visualization of protein interactions in living plant cells using bimolecular fluorescence complementationTHE PLANT JOURNAL, Issue 3 2004Michael Walter Summary Dynamic networks of protein,protein interactions regulate numerous cellular processes and determine the ability to respond appropriately to environmental stimuli. However, the investigation of protein complex formation in living plant cells by methods such as fluorescence resonance energy transfer has remained experimentally difficult, time consuming and requires sophisticated technical equipment. Here, we report the implementation of a bimolecular fluorescence complementation (BiFC) technique for visualization of protein,protein interactions in plant cells. This approach relies on the formation of a fluorescent complex by two non-fluorescent fragments of the yellow fluorescent protein brought together by association of interacting proteins fused to these fragments (Hu et al., 2002). To enable BiFC analyses in plant cells, we generated different complementary sets of expression vectors, which enable protein interaction studies in transiently or stably transformed cells. These vectors were used to investigate and visualize homodimerization of the basic leucine zipper (bZIP) transcription factor bZIP63 and the zinc finger protein lesion simulating disease 1 (LSD1) from Arabidopsis as well as the dimer formation of the tobacco 14-3-3 protein T14-3c. The interaction analyses of these model proteins established the feasibility of BiFC analyses for efficient visualization of structurally distinct proteins in different cellular compartments. Our investigations revealed a remarkable signal fluorescence intensity of interacting protein complexes as well as a high reproducibility and technical simplicity of the method in different plant systems. Consequently, the BiFC approach should significantly facilitate the visualization of the subcellular sites of protein interactions under conditions that closely reflect the normal physiological environment. [source] Assessment of physiological conditions in E. coli fermentations by epifluorescent microscopy and image analysisBIOTECHNOLOGY PROGRESS, Issue 3 2009Sónia Carneiro Abstract The development of monitoring methods for assessing the physiological state of microorganisms during recombinant fermentation processes has been encouraged by the need to evaluate the influence of processing conditions in recombinant protein production. In this work, a technique based on microscopy and image analysis was developed that allows the simultaneous quantification of parameters associated with viability and fluorescent protein production in recombinant Escherichia coli fermentations. Images obtained from light microscopy with phase contrast are used to assess the total number of cells in a given sample and, from epifluorescence microscopy, both protein producing and injured cells are evaluated using two different fluorochromes: propidium iodide and enhanced yellow fluorescent protein. This technique revealed the existence of different cell populations in the recombinant E. coli fermentation broth that were evaluated along four batch fermentations, complementing information obtained with standard techniques to study the effects of the temperature and induction time in recombinant protein production processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] pH-insensitive glucose indicatorsBIOTECHNOLOGY PROGRESS, Issue 5 2008Jared R. Garrett Abstract There is an urgent need for developing a biosensor that can real-time and noninvasively determine glucose concentration within living cells. In our previous study, we have engineered a glucose indicator protein (GIP) that can provide continuous glucose monitoring through a conformation change-induced Förster resonance-energy transfer measurement. Because of the pH-sensitivity of the fluorescent proteins used in the GIP construction, the GIP made from these fluorescent proteins is less tolerant to a pH change, especially to the acidic environment. It has been well documented that intracellular pH does not always remain the same, and it fluctuates in metabolism and other cellular activities and also differs between cellular compartments. To address these issues, we developed a GIP that can tolerate to pH change. This GIP was constructed by flanking a glucose binding protein with a cyan fluorescent protein and a pH-insensitive yellow fluorescent protein. Our experimental results indicated that the new GIP is more tolerant to pH change. The glucose response of this new GIP kept almost unchanged from pH 7.3 to 5.3, suggesting its capability of tolerating to acidic environment. This capability is desirable for intracellular glucose measurement. [source] Monitoring Protein Interactions in the Living Cell Through the Fluorescence Decays of the Cyan Fluorescent ProteinCHEMPHYSCHEM, Issue 7 2006Regis Grailhe Dr. Abstract Using fluorescence lifetime microspectroscopy and imaging techniques, we have studied the fluorescence of cyan fluorescent protein (CFP) transiently expressed in HEK-293 cells, in the presence or absence of its fluorescence resonance energy transfer (FRET) partner, yellow fluorescent protein (YFP). When the two proteins are attached through a 27-amino-acid linker, a 33,% average efficiency of intramolecular energy transfer is accurately determined inside the cell. Additionally, we observe a systematic quenching of the CFP fluorescence with increasing levels of protein expression. This quenching cannot be accounted for by formation of the previously described dimer of GFP-related proteins, since its magnitude is unchanged when the fluorescent proteins carry the mutation A206K shown to dissociate this dimer in vitro. Even when the intracellular protein concentration largely exceeds the in vitro dissociation constant of the dimer, self-association remains undetectable, either between free proteins or intramolecularly within the CFP,YFP construct. Instead, the detailed concentration effects are satisfactorily accounted for by a model of intermolecular, concentration-dependent energy transfer, arising from molecular proximity and crowding. In the case of CFP alone, we suggest that self-quenching could result from a pseudo-homo FRET mechanism between different, spectrally shifted emissive forms of the protein. These phenomena require careful consideration in intracellular FRET studies. [source] Fluorescence resonance energy transfer and anisotropy reveals both hetero- and homo-energy transfer in the pleckstrin homology-domain and the parathyroid hormone-receptorMICROSCOPY RESEARCH AND TECHNIQUE, Issue 1 2009Ralf Steinmeyer Abstract We present a method and an apparatus of polarized fluorescence resonance energy transfer (FRET) and anisotropy imaging microscopy done in parallel for improved interpretation of the photophysical interactions. We demonstrate this apparatus to better determine the protein,protein interactions in the pleckstrin homology domain and the conformational changes in the Parathyroid Hormone Receptor, a G-protein coupled receptor, both fused to the cyan and yellow fluorescent proteins for either inter- or intramolecular FRET. In both cases, the expression levels of proteins and also background autofluorescence played a significant role in the depolarization values measured in association with FRET. The system has the sensitivity and low-noise capability of single-fluorophore detection. Using counting procedures from single-molecule methods, control experiments were performed to determine number densities of green fluorescence protein variants CFP and YFP where homo resonance energy transfer can occur. Depolarization values were also determined for flavins, a common molecule of cellular background autofluorescence. From the anisotropy measurements of donor and acceptor, the latter when directly excited or when excited by energy transfer, we find that our instrumentation and method also characterizes crucial effects from homotransfer, polarization specific photobleaching and background molecules. Microsc. Res. Tech., 2009. © 2008 Wiley-Liss, Inc. [source] | ||||||||||||||||